One objective of this research project is to apply a newly developed "dual spin probe method" for the measurement of electrical surface potentials to study the charge distributions at both aqueous interfaces of inner mitochondrial membranes as a function of their state of energization. The distribution of surface charge among lipid and protein components of the membrane will also be determined by this method and by studying the membrane binding of cytochrome c labeled with a paramagnetic imidoester reagent. The second objective of the research is to characterize the order and microviscosity profile across the inner mitochondrial membrane including the aqueous interface domains. The interaction of spin probes with redox components of the electron transport chain will also be studied by exploiting spin probe reduction within defined membrane regions.